1. Field of the Invention
The present invention relates to a method for peptide C-terminal fragment sequence analysis wherein fragments are cleaved and collected from the carboxyl terminus (hereinafter referred to as the C-terminus) of a protein or peptide and subsequently analyzed for amino acid sequence, and to an apparatus for collecting a peptide fragment which can be used in the peptide fragment cleavage and collection process.
2. Discussion of the Related Art
Conventional methods for peptide C-terminal analysis include the hydrazinolysis method and the carboxypeptidase method. The carboxypeptidase method, based on the action of various kinds of carboxypeptidase to sequentially break peptide bonds from the carboxyl terminus of the polypeptide, has some drawbacks, such as complex operation, the requirement for a relatively large sample volume, and unreliable results of sequencing.
As a modified method free from these drawbacks, Japanese Patent Laid-Open No. 235600/1989 discloses a method wherein a peptide is cleaved with Lys-C specific cleavage enzyme, each resulting fragment is bound to a solid support having a functional group capable of reacting with the .alpha.-amino group and .epsilon.-amino group of the fragment to form a covalent bond, then the peptide bond between the .alpha.-amino terminal amino acid residue of each fragment and the amino acid residue adjacent thereto is cleaved by acid treatment, and a C-terminal peptide in a free form is collected. The same publication also discloses that the C-terminal peptide thus obtained can be used for amino acid sequence analysis by various methods of amino acid sequence analysis.
However, when this method is used for fragment collection, there are still several problems in the analysis of the amino acid sequence. Specifically, it is well-known that hydrophobic peptides of relatively short chains consisting of not more than about several 10 amino acids, particularly peptides having a hydrophobic amino acid at the C-terminus, are difficult to trap with polybrene or other polyvalent ion carriers used in the gas-phase protein sequencer, and sample wash-out is therefore likely to occur, which hampers the completion of amino acid sequence analysis.
Also, there has been no suitable peptide fragment collector developed for this process. Conventional collectors are configured, for example, as illustrated in FIG. 3. Specifically, reagents 31 and 32 are added to a sample loaded in a reactor (column) R to carry out cleavage or decomposition of the sample in the reactor, and the cleaved (decomposed) sample is dispensed to a recovery bottle 12 or a waste liquid bottle 13, both provided in the downstream of the reactor R.
In this case, switching between the recovery bottle 12 and the waste liquid bottle 13 is achieved by the operation of valve V. In FIG. 3, V1 represents a two-way valve and V2 and V3 respectively represent a three-way valve. Reagents 31 and 32 are supplied by the pressure of inert gas. In conventional collectors as exemplified above, the dispensing of the sample is achieved by valve operation and, therefore, contamination can occur when there is a dead volume in a valve.
Also, when using a reagent or a sample which precipitates upon drying, such as urea, it is undesirable to pass it through valves because precipitation occurs in the valves. Such limitative factors make it impossible to carry out a peptide cleaving reaction and collect peptide fragments using such conventional collectors.
Furthermore, when the reactor is packed with a packing material, the packing material can escape and cause damage downstream to the valves.